Metastasis to the regional lymph nodes occurs in about 60% of patients with squamous cell carcinoma of the head and neck (SCCHN) [1] and is the most important indicator of disease prognosis and outcome. A strategy is needed to identify patients whose cancers are likely to be metastatic, which will allow for early interventions that may prevent metastasis and thereby improve survival and quality of life in SCCHN patients. The goal of this proposal is to develop PET and optical imaging agents to quantify integrin (also called very late antigen 4, or VLA-4) and CXC chemokine receptor 4 (CXCR4) levels, both of which play a putative role in the metastasis of head and neck cancer to lymph nodes. Tumor lymphangiogenesis is the growth of new lymphatic vessels within the periphery of the tumor, and this has been correlated to the formation of lymph node metastases. Tumor associated macrophages (TAMs) have been shown to play important roles in primary tumor growth and metastasis, including promoting tumor angiogenesis, tumor invasion at the edge, tumor cell intravasation into the blood stream and lung colonization. Lymphatic endothelial cells and TAMs are both found to express high levels of integrin, and one of the goals of this proposal is to image these two pathways collectively. The CXCR4/SDF-1 signaling pathway has also been found to serve an important role in the promotion of cancer metastasis. The hypothesis to be addressed in this proposal is that the levels of integrin in lymphatic endothelial cells and tumor associated macrophages (TAMs), and CXCR4 in primary SCCHN tumors as determined by PET and optical imaging, will correlate with metastasis of the primary tumor to the lymph nodes in orthotopic mouse models of SCCHN. To address the hypothesis we will investigate integrin -targeting PET and optical agents for imaging lymphangiogenesis and TAMs collectively, and CXCR4- targeting agents for imaging chemokine levels in primary tumors. Two orthotopic mouse models of SCCHN, where either highly metastatic (OSC-19) or non-metastatic (UM22B) SCCHN tumor cells are injected into the floor of the mouth. PET and optical Imaging data will be quantified and correlated with rates and extent of metastasis. Immunohistochemistry and flow cytometry data will be obtained to validate levels of integrin and CXCR4 from excised tumors. At the end of the 2-year funding period, we anticipate that there will be evidence for the imaging of lymphangiogenesis and TAMs through VLA-4 targeting and CXCR4 expression in mouse models of SCCHN and correlation of metastatic potential with uptake of the targeted imaging agents. Ultimately, the global question to be addressed is whether SCCHN patients with primary tumors demonstrating tumor lymphangiogenesis, high levels of TAMs and/or high CXCR4 expression will predict the risk for metastasis before their cancer spreads, so that the appropriate interventions can be applied.